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Increasing the ability of chemotherapeutic drugs to kill cancer cells is often hampered by a limited understanding of their mechanism of action. Camptothecins, such as topotecan, induce cell death by poisoning DNA topoisomerase I, an enzyme capable of removing DNA supercoils. Topotecan is thought to stabilize a covalent topoisomerase-DNA complex, rendering it an obstacle to DNA replication forks. Here we use single-molecule nanomanipulation to monitor the dynamics of human topoisomerase I in the presence of topotecan. This allowed us to detect the binding and unbinding of an individual topotecan molecule in real time and to quantify the drug-induced trapping of topoisomerase on DNA. Unexpectedly, our findings also show that topotecan significantly hinders topoisomerase-mediated DNA uncoiling, with a more pronounced effect on the removal of positive (overwound) versus negative supercoils. In vivo experiments in the budding yeast verified the resulting prediction that positive supercoils would accumulate during transcription and replication as a consequence of camptothecin poisoning of topoisomerase I. Positive supercoils, however, were not induced by drug treatment of cells expressing a catalytically active, camptothecin-resistant topoisomerase I mutant. This combination of single-molecule and in vivo data suggests a cytotoxic mechanism for camptothecins, in which the accumulation of positive supercoils ahead of the replication machinery induces potentially lethal DNA lesions.

Resistance to current chemotherapeutic or radiation-based cancer treatment strategies is a serious concern. Cancer stem cells (CSCs) are typically able to evade treatment and establish a recurrent tumor or metastasis, and it is these that lead to the majority of cancer deaths. Therefore, a major current goal is to develop treatment strategies that eliminate the resistant CSCs as well as the bulk tumor cells in order to achieve complete disease clearance. Aldehyde dehydrogenases (ALDHs) are important for maintenance and differentiation of stem cells as well as normal development. There is expanding evidence that ALDH expression increases in response to therapy and promotes chemoresistance and survival mechanisms in CSCs. This perspective will discuss a paper by

ObjectiveAldehyde dehydrogenase (ALDH) expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance.MethodsIsogenic ovarian cancer cell lines for platinum sensitivity (A2780) and platinum resistant (A2780/CP70) as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators.ResultsALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDHHIGH displayed significantly lower progression free survival than the patients with ALDHLOW cells (9 vs. 3 months, respectively p<0.01). ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ) and replication checkpoint (pS317 Chk1) were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells.ConclusionThis data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.